LncRNA-HOTAIR/MEG3通过自噬调控角质形成细胞分化在银屑病发病中的机制研究

Much evidence shows the presence of differentiation abnormalities of keratinocyte (KC) in psoriasis patients. However, whether the deregulation of LncRNA-autophagy axis plays important role during such process is still unclear. Therefore, we suppose a new mechanism that the dysfunction of LncRNA-HOTAIR/MEG3 induced autophagy in keratinocyte may be an important cause of the differentiation abnormalities. In our study, we first aim to detect the expression of LncRNA-HOTAIR/MEG3 in skin tissues and peripheral blood CD4+ T cells of psoriasis patients, in order to analyze the correlation between their expression and the severity of psoriasis among patients. In addition, we will explore the role LncRNA-HOTAIR/MEG3 play in autophagy and differentiation of KC by conducting overexpression/knockdown of LncRNA-HOTAIR/MEG3 in KC in vitro. We will further verify the function of LncRNA-HOTAIR/MEG3-autophagy-differentiation axis in the pathogenesis of psoriasis by constructing psoriasis-like lesions in LncRNA-HOTAIR/MEG3 knocked out mice as well as the control group. Finally, by performing a series of experiments including RNA pull down, mass spectrometry, overexpression/knockdown of RNA-binding proteins in KC, we will investigate the the mechanism that how LncRNA-HOTAIR/MEG3 regulate autophagy in KC. This study will helps us to further reveal the pathogenesis of psoriasis.

银屑病中存在角质形成细胞（KC）分化（角化）异常，然而lncRNA-自噬调控机制是否参与这种异常并不清楚。本课题提出LncRNA-HOTAIR/MEG3通过自噬调控KC分化参与了银屑病的发病这一科学假设。本研究首先分析银屑病患者皮损和外周血CD4+T细胞中LncRNA-HOTAIR/MEG3的表达水平，并分析其病情的相关性；进而，在体外培养的KC中通过基因敲低和过表达的手段，分析LncRNA-HOTAIR/MEG3其对细胞自噬和分化的影响，并在LncRNA-HOTAIR/MEG3敲除的小鼠银屑病模型中验证LncRNA-HOTAIR/MEG3-自噬-细胞分化这一分子机制对银屑病发病的作用；最后，在体外培养的KC中通过RNA pull down、质谱分析、RNA结合蛋白的基因敲低或过表达等方法，阐明LncRNA-HOTAIR/MEG3调控自噬的分子机制。本研究有助于阐明银屑病的发病机制。

项目的背景.正常角质形成细胞内基础自噬水平较高，其对于维持角质形成细胞的正常功能具有重要意义。同时，细胞自噬又与多种调节性细胞死亡模式（RCDs）存在着密切的相互联系。..主要研究内容.本项目中，我们在自噬关键基因ATG7角质形成细胞条件性敲除小鼠（cKO）及背景小鼠中构建了咪喹莫特诱导的银屑病样皮炎模型，并比对分析了二者的炎症表型差异，检测了其对于表皮角质形成细胞增殖、分化的影响；构建了ATG7敲低的角质形成细胞系HaCaT细胞，通过一系列体外实验探究ATG7在银屑病发病过程中对于角质形成细胞增殖、分化、炎症的影响。另外，我们在银屑病模型中探索了gasdermin D介导的细胞焦亡和细胞铜死亡对于其发病的影响。在变应性接触性皮炎（ACD）中，探讨了MLKL介导的程序性坏死诱发的关键炎症介质HMGB1的分泌对炎症反应的自循环放大效应。..重要结果和关键数据.ATG7 cKO小鼠肉眼及组织病理学表型均较背景鼠进一步加剧；ATG7敲降HaCaT细胞系与对照组细胞暴露于银屑病样混合炎症因子环境中时，ATG7敲降HaCaT细胞较对照细胞表现出更显著的增殖加速与分化障碍；一系列银屑病相关信号通路活化和炎症因子转录上调在ATG7敲降HaCaT细胞中更为明显。银屑病角质形成细胞出现异常的gasdermin D介导的细胞焦亡介导了银屑病表皮角质形成细胞的异常增殖、异常分化和异常的Th17型免疫应答，靶向gasdermin D介导的细胞焦亡可以呈现显著的干预银屑病的效应；银屑病发病过程中铜死亡的异常缺失可通过减少皮肤微环境中的乳酸含量加剧银屑病发病，诱导角质形成细胞铜死亡可增加皮损微环境内的乳酸水平，从而调节Treg / Th17免疫稳态来缓解银屑病。MLKL介导的角质形成细胞程序性坏死在ACD发病过程中发挥着关键效应，抑制这一效应可通过减少其HMGB1的释放来阻断中细粒细胞的募集和激活，从而缓解皮肤炎症。..科学意义.角质形成细胞自噬与其他RCDs的异常改变在银屑病等炎症性皮肤病发病过程中起着重要作用。靶向异常的调节性细胞死亡有望成为银屑病等炎症性皮肤病的潜在诊疗策略。